Human cytomegalovirus (HCMV) persistently infects over half of the Veteran population and causes disease and death in a sizeable segment of this population despite advances in antiviral therapy. Novel strategies to mitigate the disease burden resulting from HCMV infection are needed. The HCMV MIE enhancer/promoter serves as the lytic switch for the virus. It is turned on to initiate the viral replicative cycle and turned off during viral latency. Knowledge about the ways in which the HCMV MIE lytic switch is controlled has the potential to inform the development of new therapies to pre-empt viral replication. Our laboratory is at the forefront of investigations into defining the control mechanisms. These early results have led us to hypothesize that the transcriptional regulatory hierarchies and cis-regulatory codes in control of HCMV MIE lytic switch activation differ between cell types in acutely productive and reactivation infections. Preliminary data in human pluripotent N-Tera2 cells (NT2) suggest that HCMV MIE reactivation is best achieved by a combination of different cues and multiple regulatory pathways. There is both a need to further elucidate the cues and regulatory pathways involved and a need to evaluate these findings in a HCMV latency model in dendritic- monocytic cell (D/M) precursors. These results will be the first to elucidate the molecular mechanisms controlling HCMV MIE lytic switch reactivation. Preliminary data in acutely infected permissive fibroblasts reveal a novel regulatory mechanism which functions atypically in the post-immediate-early phase of infection and acts together with another yet unidentified regulatory mechanism. Filling in the gaps in knowledge about action, mechanism, and purpose of this regulatory mechanism is anticipated to shift paradigm in explaining how the MIE enhancer governs HCMV replication. The specific aims of this project are to delineate the differential roles of specific transcription factor pathways and cis-regulatory codes in HCMV MIE lytic switch activation in: 1) acutely infected permissive fibroblast, endothelial, epithelial, and myeloid cells; and 2) quiescently infected NT2 and D/M precursors. The research plan will draw on both innovative approaches and tools. The use of advanced technology and state-of-the-art methods makes feasible the detailing of these pathways at unparalleled depth in diverse cellular settings of biological import. An outstanding research environment and an accomplished research team that interfaces well with the Central Microscopy Research, Proteomics, and Flow Cytometry facilities at the University of Iowa are ideally suited for carrying out this plan. The discovery of viral regulatory mechanisms that can be disrupted to stop HCMV gene expression and replication before it begins potentially provides a new direction in antiviral drug development, with the goal of improving Veterans' health.